Fossil-calibrated molecular clock data enable reconstruction of steps leading to differentiated multicellularity and anisogamy in the Volvocine algae.

BACKGROUND: Throughout its nearly four-billion-year history, life has undergone evolutionary transitions in which simpler subunits have become integrated to form a more complex whole. Many of these transitions opened the door to innovations that resulted in increased biodiversity and/or organismal efficiency. The evolution of multicellularity from unicellular forms represents one such transition, one that paved the way for cellular differentiation, including differentiation of male and female gametes. A useful model for studying the evolution of multicellularity and cellular differentiation is the volvocine algae, a clade of freshwater green algae whose members range from unicellular to colonial, from undifferentiated to completely differentiated, and whose gamete types can be isogamous, anisogamous, or oogamous. To better understand how multicellularity, differentiation, and gametes evolved in this group, we used comparative genomics and fossil data to establish a geologically calibrated roadmap of when these innovations occurred. RESULTS: Our ancestral-state reconstructions, show that multicellularity arose independently twice in the volvocine algae. Our chronograms indicate multicellularity evolved during the Carboniferous-Triassic periods in Goniaceae + Volvocaceae, and possibly as early as the Cretaceous in Tetrabaenaceae. Using divergence time estimates we inferred when, and in what order, specific developmental changes occurred that led to differentiated multicellularity and oogamy. We find that in the volvocine algae the temporal sequence of developmental changes leading to differentiated multicellularity is much as proposed by David Kirk, and that multicellularity is correlated with the acquisition of anisogamy and oogamy. Lastly, morphological, molecular, and divergence time data suggest the possibility of cryptic species in Tetrabaenaceae. CONCLUSIONS: Large molecular datasets and robust phylogenetic methods are bringing the evolutionary history of the volvocine algae more sharply into focus. Mounting evidence suggests that extant species in this group are the result of two independent origins of multicellularity and multiple independent origins of cell differentiation. Also, the origin of the Tetrabaenaceae-Goniaceae-Volvocaceae clade may be much older than previously thought. Finally, the possibility of cryptic species in the Tetrabaenaceae provides an exciting opportunity to study the recent divergence of lineages adapted to live in very different thermal environments.

Spontaneous Emergence of Multicellular Heritability.

The major transitions in evolution include events and processes that result in the emergence of new levels of biological individuality. For collectives to undergo Darwinian evolution, their traits must be heritable, but the emergence of higher-level heritability is poorly understood and has long been considered a stumbling block for nascent evolutionary transitions. Using analytical models, synthetic biology, and biologically-informed simulations, we explored the emergence of trait heritability during the evolution of multicellularity. Prior work on the evolution of multicellularity has asserted that substantial collective-level trait heritability either emerges only late in the transition or requires some evolutionary change subsequent to the formation of clonal multicellular groups. In a prior analytical model, we showed that collective-level heritability not only exists but is usually more heritable than the underlying cell-level trait upon which it is based, as soon as multicellular groups form. Here, we show that key assumptions and predictions of that model are borne out in a real engineered biological system, with important implications for the emergence of collective-level heritability.

Green Algal Models for Multicellularity.

The repeated evolution of multicellularity across the tree of life has profoundly affected the ecology and evolution of nearly all life on Earth. Many of these origins were in different groups of photosynthetic eukaryotes, or algae. Here, we review the evolution and genetics of multicellularity in several groups of green algae, which include the closest relatives of land plants. These include millimeter-scale, motile spheroids of up to 50,000 cells in the volvocine algae; decimeter-scale seaweeds in the genus Ulva (sea lettuce); and very plantlike, meter-scale freshwater algae in the genus Chara (stoneworts). We also describe algae in the genus Caulerpa, which are giant, multinucleate, morphologically complex single cells. In each case, we review the life cycle, phylogeny, and genetics of traits relevant to the evolution of multicellularity, and genetic and genomic resources available for the group in question. Finally, we suggest routes toward developing these groups as model organisms for the evolution of multicellularity.

Phylotranscriptomics points to multiple independent origins of multicellularity and cellular differentiation in the volvocine algae.

BACKGROUND: The volvocine algae, which include the single-celled species Chlamydomonas reinhardtii and the colonial species Volvox carteri, serve as a model in which to study the evolution of multicellularity and cellular differentiation. Studies reconstructing the history of this group have by and large relied on datasets of one to a few genes for phylogenetic inference and ancestral character state reconstruction. As a result, volvocine phylogenies lack concordance depending on the number and/or type of genes (i.e., chloroplast vs nuclear) chosen for phylogenetic inference. While multiple studies suggest that multicellularity evolved only once in the volvocine algae, that each of its three colonial families is monophyletic, and that there have been at least three independent origins of cellular differentiation in the group, other studies call into question one or more of these conclusions. An accurate assessment of the evolutionary history of the volvocine algae requires inference of a more robust phylogeny. RESULTS: We performed RNA sequencing (RNA-seq) on 55 strains representing 47 volvocine algal species and obtained similar data from curated databases on 13 additional strains. We then compiled a dataset consisting of transcripts for 40 single-copy, protein-coding, nuclear genes and subjected the predicted amino acid sequences of these genes to maximum likelihood, Bayesian inference, and coalescent-based analyses. These analyses show that multicellularity independently evolved at least twice in the volvocine algae and that the colonial family Goniaceae is not monophyletic. Our data further indicate that cellular differentiation arose independently at least four, and possibly as many as six times, within the volvocine algae. CONCLUSIONS: Altogether, our results demonstrate that multicellularity and cellular differentiation are evolutionarily labile in the volvocine algae, affirming the importance of this group as a model system for the study of major transitions in the history of life.

Evolution of altruistic cooperation among nascent multicellular organisms.

Cooperation is a classic solution to hostile environments that limit individual survival. In extreme cases this may lead to the evolution of new types of biological individuals (e.g., eusocial super-organisms). We examined the potential for interindividual cooperation to evolve via experimental evolution, challenging nascent multicellular "snowflake yeast" with an environment in which solitary multicellular clusters experienced low survival. In response, snowflake yeast evolved to form cooperative groups composed of thousands of multicellular clusters that typically survive selection. Group formation occurred through the creation of protein aggregates, only arising in strains with high (>2%) rates of cell death. Nonetheless, it was adaptive and repeatable, although ultimately evolutionarily unstable. Extracellular protein aggregates act as a common good, as they can be exploited by cheats that do not contribute to aggregate production. These results highlight the importance of group formation as a mechanism for surviving environmental stress, and underscore the remarkable ease with which even simple multicellular entities may evolve-and lose-novel social traits.

De novo origins of multicellularity in response to predation.

The transition from unicellular to multicellular life was one of a few major events in the history of life that created new opportunities for more complex biological systems to evolve. Predation is hypothesized as one selective pressure that may have driven the evolution of multicellularity. Here we show that de novo origins of simple multicellularity can evolve in response to predation. We subjected outcrossed populations of the unicellular green alga Chlamydomonas reinhardtii to selection by the filter-feeding predator Paramecium tetraurelia. Two of five experimental populations evolved multicellular structures not observed in unselected control populations within ~750 asexual generations. Considerable variation exists in the evolved multicellular life cycles, with both cell number and propagule size varying among isolates. Survival assays show that evolved multicellular traits provide effective protection against predation. These results support the hypothesis that selection imposed by predators may have played a role in some origins of multicellularity.

Trait heritability in major transitions.

BACKGROUND: Increases in biological complexity and the origins of life's hierarchical organization are described by the "major transitions" framework. A crucial component of this paradigm is that after the transition in complexity or organization, adaptation occurs primarily at the level of the new, higher-level unit. For collective-level adaptations to occur, though, collective-level traits-properties of the group, such as collective size-must be heritable. Since collective-level trait values are functions of lower-level trait values, collective-level heritability is related to particle-level heritability. However, the nature of this relationship has rarely been explored in the context of major transitions. RESULTS: We examine relationships between particle-level heritability and collective-level heritability for several functions that express collective-level trait values in terms of particle-level trait values. For clonal populations, when a collective-level trait value is a linear function of particle-level trait values and the number of particles per collective is fixed, the heritability of a collective-level trait is never less than that of the corresponding particle-level trait and is higher under most conditions. For more complicated functions, collective-level heritability is higher under most conditions, but can be lower when the environment experienced by collectives is heterogeneous. Within-genotype variation in collective size reduces collective-level heritability, but it can still exceed particle-level heritability when phenotypic variance among particles within collectives is large. These results hold for a diverse sample of biologically relevant traits. CONCLUSIONS: Rather than being an impediment to major transitions, we show that, under a wide range of conditions, the heritability of collective-level traits is actually higher than that of the corresponding particle-level traits. High levels of collective-level trait heritability thus arise "for free," with important implications not only for major transitions but for multilevel selection in general.

Genetics of a de novo origin of undifferentiated multicellularity.

The evolution of multicellularity was a major transition in evolution and set the stage for unprecedented increases in complexity, especially in land plants and animals. Here, we explore the genetics underlying a de novo origin of multicellularity in a microbial evolution experiment carried out on the green alga Chlamydomonas reinhardtii. We show that large-scale changes in gene expression underlie the transition to a multicellular life cycle. Among these, changes to genes involved in cell cycle and reproductive processes were overrepresented, as were changes to C. reinhardtii-specific and volvocine-specific genes. These results suggest that the genetic basis for the experimental evolution of multicellularity in C. reinhardtii has both lineage-specific and shared features, and that the shared features have more in common with C. reinhardtii's relatives among the volvocine algae than with other multicellular green algae or land plants.

Multicellularity Drives the Evolution of Sexual Traits.

From the male peacock's tail plumage to the floral displays of flowering plants, traits related to sexual reproduction are often complex and exaggerated. Why has sexual reproduction become so complicated? Why have such exaggerated sexual traits evolved? Early work posited a connection between multicellularity and sexual traits such as anisogamy (i.e., the evolution of small sperm and large eggs). Anisogamy then drives the evolution of other forms of sexual dimorphism. Yet the relationship between multicellularity and the evolution of sexual traits has not been empirically tested. Given their extensive variation in both multicellular complexity and sexual systems, the volvocine green algae offer a tractable system for understanding the interrelationship of multicellular complexity and sex. Here we show that species with greater multicellular complexity have a significantly larger number of derived sexual traits, including anisogamy, internal fertilization, and secondary sexual dimorphism. Our results demonstrate that anisogamy repeatedly evolved from isogamous multicellular ancestors and that anisogamous species are larger and produce larger zygotes than isogamous species. In the volvocine algae, the evolution of multicellularity likely drives the evolution of anisogamy, and anisogamy subsequently drives secondary sexual dimorphism. Multicellularity may set the stage for the overall diversity of sexual complexity throughout the Tree of Life.

Analysis of motility in multicellular Chlamydomonas reinhardtii evolved under predation.

The advent of multicellularity was a watershed event in the history of life, yet the transition from unicellularity to multicellularity is not well understood. Multicellularity opens up opportunities for innovations in intercellular communication, cooperation, and specialization, which can provide selective advantages under certain ecological conditions. The unicellular alga Chlamydomonas reinhardtii has never had a multicellular ancestor yet it is closely related to the volvocine algae, a clade containing taxa that range from simple unicells to large, specialized multicellular colonies. Simple multicellular structures have been observed to evolve in C. reinhardtii in response to predation or to settling rate-based selection. Structures formed in response to predation consist of individual cells confined within a shared transparent extracellular matrix. Evolved isolates form such structures obligately under culture conditions in which their wild type ancestors do not, indicating that newly-evolved multicellularity is heritable. C. reinhardtii is capable of photosynthesis, and possesses an eyespot and two flagella with which it moves towards or away from light in order to optimize input of radiant energy. Motility contributes to C. reinhardtii fitness because it allows cells or colonies to achieve this optimum. Utilizing phototaxis to assay motility, we determined that newly evolved multicellular strains do not exhibit significant directional movement, even though the flagellae of their constituent unicells are present and active. In C. reinhardtii the first steps towards multicellularity in response to predation appear to result in a trade-off between motility and differential survivorship, a trade-off that must be overcome by further genetic change to ensure long-term success of the new multicellular organism.

Repeated evolution and reversibility of self-fertilization in the volvocine green algae.

Outcrossing and self-fertilization are fundamental strategies of sexual reproduction, each with different evolutionary costs and benefits. Self-fertilization is thought to be an evolutionary "dead-end" strategy, beneficial in the short term but costly in the long term, resulting in self-fertilizing species that occupy only the tips of phylogenetic trees. Here, we use volvocine green algae to investigate the evolution of self-fertilization. We use ancestral-state reconstructions to show that self-fertilization has repeatedly evolved from outcrossing ancestors and that multiple reversals from selfing to outcrossing have occurred. We use three phylogenetic metrics to show that self-fertilization is not restricted to the tips of the phylogenetic tree, a finding inconsistent with the view of self-fertilization as a dead-end strategy. We also find no evidence for higher extinction rates or lower speciation rates in selfing lineages. We find that self-fertilizing species have significantly larger colonies than outcrossing species, suggesting the benefits of selfing may counteract the costs of increased size. We speculate that our macroevolutionary results on self-fertilization (i.e., non-tippy distribution, no decreased diversification rates) may be explained by the haploid-dominant life cycle that occurs in volvocine algae, which may alter the costs and benefits of selfing.

Rediscovery of the species of 'ancestral Volvox': morphology and phylogenetic position of Pleodorina sphaerica (Volvocales, Chlorophyceae) from Thailand.

Pleodorina sphaerica Iyengar was considered to be a phylogenetic link between Volvox and the type species Pleodorina californica Shaw because it has small somatic cells distributed from the anterior to posterior poles in 64- or 128-celled vegetative colonies. However, cultural studies and molecular and ultrastructural data are lacking in P. sphaerica, and this species has not been recorded since 1951. Here, we performed light and electron microscopy and molecular phylogeny of P. sphaerica based on newly established culture strains originating from Thailand. Morphological features of the present Thai species agreed well with those of the previous studies of the Indian material of P. sphaerica and with those of the current concept of the advanced members of the Volvocaceae. The present P. sphaerica strains exhibited homothallic sexuality; male and facultative female colonies developed within a single clonal culture. Chloroplast multigene phylogeny demonstrated that P. sphaerica was sister to two other species of Pleodorina (P. californica and Pleodorina japonica Nozaki) without posterior somatic cells, and these three species of Pleodorina formed a robust clade, which was positioned distally in the large monophyletic group including nine taxa of Volvox sect. Merrillosphaera and Volvox (sect. Janetosphaera) aureus Ehrenberg. Based on the present phylogenetic results, evolutionary losses of posterior somatic cells might have occurred in the ancestor of P. californica and P. japonica. Thus, P. sphaerica might represent an ancestral morphology of Pleodorina, rather than of Volvox.

Origins of multicellular complexity: Volvox and the volvocine algae.

The collection of evolutionary transformations known as the 'major transitions' or 'transitions in individuality' resulted in changes in the units of evolution and in the hierarchical structure of cellular life. Volvox and related algae have become an important model system for the major transition from unicellular to multicellular life, which touches on several fundamental questions in evolutionary biology. The Third International Volvox Conference was held at the University of Cambridge in August 2015 to discuss recent advances in the biology and evolution of this group of algae. Here, I highlight the benefits of integrating phylogenetic comparative methods and experimental evolution with detailed studies of developmental genetics in a model system with substantial genetic and genomic resources. I summarize recent research on Volvox and its relatives and comment on its implications for the genomic changes underlying major evolutionary transitions, evolution and development of complex traits, evolution of sex and sexes, evolution of cellular differentiation and the biophysics of motility. Finally, I outline challenges and suggest future directions for research into the biology and evolution of the volvocine algae.

Fitness and Individuality in Complex Life Cycles.

Complex life cycles are common in the eukaryotic world, and they complicate the question of how to define individuality. Using a bottom-up, gene-centric approach, I consider the concept of fitness in the context of complex life cycles. I analyze the fitness effects of an allele (or a trait) on different biological units within a complex life history and how these effects drive evolutionary change within populations. Based on these effects, I attempt to construct a concept of fitness that accurately predicts evolutionary change in the context of complex life cycles.

Fitness trade-offs and developmental constraints in the evolution of soma: an experimental study in a volvocine alga.

BACKGROUND: The evolution of mortal somatic cells was a critical step in the evolution of complex body plans and the major radiations of multicellular life. In the volvocine green algae, somatic cells are hypothesized to mitigate an increasing cost of reproduction as colony size increases, primarily by providing motility to the colony during reproduction. QUESTIONS: Does selection on colony size cause an evolutionary response in proportion of somatic cells? Does the effect of selection on colony size differ in environments that differ in the importance of motility? METHODS: We subjected an outcrossed population of the volvocine alga Pleodorina starrii to selection on colony size in still and mixed environments. After approximately 40 generations with periodic selection, we estimated the relationship between colony size and proportion of soma in evolved colonies from both environments. RESULTS: In the largest size category, colonies selected in the still environment (in which motility is hypothesized to be more important) had a higher proportion of soma than those from the mixed environment. Within-strain variation in cell number was surprisingly large: up to 16-fold for some genotypes. The positive among-species relationship between colony size and proportion of soma was paralleled within the larger (16- to 64-celled) colonies of P. starrii, but not within the smaller (4- and 8-celled) colonies, which had the highest proportions of soma, suggesting the existence of an evolutionary constraint preventing optimization of soma in the smallest size classes.

Experimental evolution of an alternating uni- and multicellular life cycle in Chlamydomonas reinhardtii.

The transition to multicellularity enabled the evolution of large, complex organisms, but early steps in this transition remain poorly understood. Here we show that multicellular complexity, including development from a single cell, can evolve rapidly in a unicellular organism that has never had a multicellular ancestor. We subject the alga Chlamydomonas reinhardtii to conditions that favour multicellularity, resulting in the evolution of a multicellular life cycle in which clusters reproduce via motile unicellular propagules. While a single-cell genetic bottleneck during ontogeny is widely regarded as an adaptation to limit among-cell conflict, its appearance very early in this transition suggests that it did not evolve for this purpose. Instead, we find that unicellular propagules are adaptive even in the absence of intercellular conflict, maximizing cluster-level fecundity. These results demonstrate that the unicellular bottleneck, a trait essential for evolving multicellular complexity, can arise rapidly via co-option of the ancestral unicellular form.

Cellular differentiation and individuality in the 'minor' multicellular taxa.

Biology needs a concept of individuality in order to distinguish organisms from parts of organisms and from groups of organisms, to count individuals and compare traits across taxa, and to distinguish growth from reproduction. Most of the proposed criteria for individuality were designed for 'unitary' or 'paradigm' organisms: contiguous, functionally and physiologically integrated, obligately sexually reproducing multicellular organisms with a germ line sequestered early in development. However, the vast majority of the diversity of life on Earth does not conform to all of these criteria. We consider the issue of individuality in the 'minor' multicellular taxa, which collectively span a large portion of the eukaryotic tree of life, reviewing their general features and focusing on a model species for each group. When the criteria designed for unitary organisms are applied to other groups, they often give conflicting answers or no answer at all to the question of whether or not a given unit is an individual. Complex life cycles, intimate bacterial symbioses, aggregative development, and strange genetic features complicate the picture. The great age of some of the groups considered shows that 'intermediate' forms, those with some but not all of the traits traditionally associated with individuality, cannot reasonably be considered ephemeral or assumed transitional. We discuss a handful of recent attempts to reconcile the many proposed criteria for individuality and to provide criteria that can be applied across all the domains of life. Finally, we argue that individuality should be defined without reference to any particular taxon and that understanding the emergence of new kinds of individuals requires recognizing individuality as a matter of degree.

Parallel evolutionary dynamics of adaptive diversification in Escherichia coli.

The causes and mechanisms of evolutionary diversification are central issues in biology. Geographic isolation is the traditional explanation for diversification, but recent theoretical and empirical studies have shown that frequency-dependent selection can drive diversification without isolation and that adaptive diversification occurring in sympatry may be an important source of biological diversity. However, there are no empirical examples in which sympatric lineage splits have been understood at the genetic level, and it is unknown how predictable this process is-that is, whether similar ecological settings lead to parallel evolutionary dynamics of diversification. We documented the genetic basis and the evolutionary dynamics of adaptive diversification in three replicate evolution experiments, in which competition for two carbon sources caused initially isogenic populations of the bacterium Escherichia coli to diversify into two coexisting ecotypes representing different physiological adaptations in the central carbohydrate metabolism. Whole-genome sequencing of clones of each ecotype from different populations revealed many parallel and some unique genetic changes underlying the derived phenotypes, including changes to the same genes and sometimes to the same nucleotide. Timelines of allele frequencies extracted from the frozen "fossil" record of the three evolving populations suggest parallel evolutionary dynamics driven at least in part by a co-evolutionary process in which mutations causing one type of physiology changed the ecological environment, allowing the invasion of mutations causing an alternate physiology. This process closely corresponds to the evolutionary dynamics seen in mathematical models of adaptive diversification due to frequency-dependent ecological interactions. The parallel genetic changes underlying similar phenotypes in independently evolved lineages provide empirical evidence of adaptive diversification as a predictable evolutionary process.

Adaptive diversification of a plastic trait in a predictably fluctuating environment.

There is substantial evidence that evolutionary diversification can occur in allopatric conditions through reduction in the degree of phenotypic plasticity when an isolated population encounters a novel, more stable environment. Plasticity is no longer favored in the new environment, either because it carries an inherent physiological cost or because it leads to production of suboptimal phenotypes. In order to explore the role of phenotypic plasticity in sympatric diversification, we modeled the ecological and evolutionary dynamics of Escherichia coli bacteria in batch cultures. Our results describe an evolutionary pathway leading to metabolic diversification in a sympatric environment without spatial structure. In an environment that fluctuates widely and predictably, evolutionary branching leads to diversification and stable coexistence of generalist and specialist ecotypes for some combinations of parameters. Diversification and stable coexistence occur when reaction norms are steep and trade-offs between metabolic pathways are convex. We conclude that, in principle, diversification due to reduced plasticity can occur without allopatric isolation, reduced environmental variability, or an explicit cost of plasticity.